Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 28, 2020; 26(20): 2570-2583
Published online May 28, 2020. doi: 10.3748/wjg.v26.i20.2570
Serum outperforms plasma in small extracellular vesicle microRNA biomarker studies of adenocarcinoma of the esophagus
Karen Chiam, George C Mayne, Tingting Wang, David I Watson, Tanya S Irvine, Tim Bright, Lorelle T Smith, Imogen A Ball, Joanne M Bowen, Dorothy M Keefe, Sarah K Thompson, Damian J Hussey
Karen Chiam, George C Mayne, Tingting Wang, David I Watson, Tanya S Irvine, Tim Bright, Lorelle T Smith, Sarah K Thompson, Damian J Hussey, Discipline of Surgery, College of Medicine and Public Health, Flinders University of South Australia, Adelaide, SA 5042, Australia
Karen Chiam, George C Mayne, Tingting Wang, David I Watson, Tanya S Irvine, Tim Bright, Lorelle T Smith, Damian J Hussey, Flinders Health and Medical Research Institute Cancer Program, College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia
Lorelle T Smith, Discipline of Surgery, The University of Adelaide, Adelaide, SA 5005, Australia
Imogen A Ball, Joanne M Bowen, Adelaide Medical School, The University of Adelaide, Adelaide, SA 5005, Australia
Dorothy M Keefe, Faculty of Health Sciences, The University of Adelaide, Adelaide, SA 5005, Australia
Author contributions: Hussey DJ conceived and supervised the study; Chiam K, Mayne GC, Watson DI, Bowen JM, Keefe DM, Thompson SK and Hussey DJ contributed to study design; Watson DI, Irvine TS, Bright T, Smith LT and White I collected patient blood samples; Wang T processed patient blood samples and performed the laboratory assays; Chiam K, Mayne GC and Hussey DJ analysed the data; Chiam K wrote the first draft of the paper; All authors contributed to revision of the manuscript in its final version.
Supported by National Health and Medical Research Council (NHMRC) Project Funding, No. APP1104281; and NHMRC Centres of Research Excellence (CRE) Grant, No. APP1040947.
Institutional review board statement: Ethical approval was obtained from the Southern Adelaide Clinical Human Research Ethics Committee and the Royal Adelaide Hospital Research Committee.
Conflict-of-interest statement: The authors have no conflict of interest to declare.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Damian J Hussey, PhD, Senior Scientist, Discipline of Surgery, College of Medicine and Public Health, Flinders University of South Australia, Bedford Park, Adelaide, SA 5042, Australia.
Received: January 1, 2020
Peer-review started: January 1, 2020
First decision: January 19, 2020
Revised: March 2, 2020
Accepted: May 13, 2020
Article in press: May 13, 2020
Published online: May 28, 2020

Circulating microRNAs (miRNAs) are potential biomarkers for many diseases. However, they can originate from non-disease specific sources, such as blood cells, and compromise the investigations for miRNA biomarkers. While small extracellular vesicles (sEVs) have been suggested to provide a purer source of circulating miRNAs for biomarkers discovery, the most suitable blood sample for sEV miRNA biomarker studies has not been defined.


To compare the miRNA profiles between matched serum and plasma sEV preparations to determine their suitability for biomarker studies.


Matched serum and plasma samples were obtained from 10 healthy controls and 10 patients with esophageal adenocarcinoma. sEV isolates were prepared from serum and plasma using ExoQuickTM and quantified using NanoSight. RNA was extracted from sEV preparations with the miRNeasy Serum/Plasma kit and profiled using the Taqman Openarray qPCR. The overall miRNA content and the expression of specific miRNAs of reported vesicular and non-vesicular origins were compared between serum and plasma sEV preparations. The diagnostic performance of a previously identified multi-miRNA biomarker panel for esophageal adenocarcinoma was also compared.


The overall miRNA content was higher in plasma sEV preparations (480 miRNAs) and contained 97.5% of the miRNAs found in the serum sEV preparations (412 miRNAs).The expression of commonly expressed miRNAs was highly correlated (Spearman’s R = 0.87, P < 0.0001) between the plasma and serum sEV preparations, but was consistently higher in the plasma sEV preparations. Specific blood-cell miRNAs (hsa-miR-223-3p, hsa-miR-451a, miR-19b-3p, hsa-miR-17-5p, hsa-miR-30b-5p, hsa-miR-106a-5p, hsa-miR-150-5p and hsa-miR-92a-3p) were expressed at 2.7 to 9.6 fold higher levels in the plasma sEV preparations compared to serum sEV preparations (P < 0.05). In plasma sEV preparations, the percentage of protein-associated miRNAs expressed at relatively higher levels (Ct 20-25) was greater than serum sEV preparations (50% vs 31%). While the percentage of vesicle-associated miRNAs expressed at relatively higher levels was greater in the serum sEV preparations than plasma sEV preparations (70% vs 44%). A 5-miRNA biomarker panel produced a higher cross validated accuracy for discriminating patients with esophageal adenocarcinoma from healthy controls using serum sEV preparations compared with plasma sEV preparations (AUROC 0.80 vs 0.54, P < 0.05).


Although plasma sEV preparations contained more miRNAs than serum sEV preparations, they also contained more miRNAs from non-vesicle origins. Serum appears to be more suitable than plasma for sEV miRNAs biomarkers studies.

Keywords: Biomarkers, Exosomes, Extracellular vesicles, Circulating microRNA, MicroRNAs, Plasma, Serum, Blood cells, Real-time polymerase chain reaction, Adenocarcinoma of esophagus

Core tip: Current evidence suggests that circulating small extracellular vesicles (sEVs) function as delivery cargo shuttles for various molecules. MicroRNAs are small non-coding RNAs with important roles in the regulation of gene expression, are often dysregulated in diseases, and are relatively stable in the circulation. MicroRNAs circulating in sEVs are consequently considered as highly suitable candidates for use as non-invasive biomarkers. Extracellular vesicle preparations derived from serum and plasma are recognised to be enriched in sEVs, but not purely comprised of them. Most circulating sEV microRNA biomarker studies have used plasma, but here we show that sEVs isolated from serum are less contaminated with blood cell and protein-associated microRNAs.